CN109336835B

CN109336835B - Fluorescent probe for detecting activity of myeloperoxidase and preparation method and application thereof

Info

Publication number: CN109336835B
Application number: CN201811333711.5A
Authority: CN
Inventors: 易涛; 刘玲燕; 魏鹏; 刘中宽; 李若涵; 曹春艳
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2018-11-09
Filing date: 2018-11-09
Publication date: 2022-10-11
Anticipated expiration: 2038-11-09
Also published as: CN109336835A

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a fluorescent probe for detecting activity of myeloperoxidase, and a preparation method and application thereof. The fluorescent probe compounds of the present invention detect myeloperoxidase activity by generating fluorescence in response to hypochlorous acid, and do not respond to other common reactive oxygen species/reactive nitrogen species. The method has the advantages of high selectivity and sensitivity on hypochlorous acid, quick response, no need of complex instruments, low detection limit on the activity of the myeloperoxidase, and capability of detecting the activity of the myeloperoxidase at the living body level. The compounds can be used as fluorescent probes in the fields of disease pre-diagnosis, fluorescence imaging and the like.

Description

Fluorescent probe for detecting activity of myeloperoxidase and preparation method and application thereof

Technical Field

The invention belongs to the technical field of analytical chemistry, and particularly relates to a fluorescent probe for detecting Myeloperoxidase (MPO) activity, and a preparation method and application thereof.

Background

Myeloperoxidase (MPO) is a class of peroxidases that uses heme as a cofactor and is present almost exclusively in bone marrow cells, two types of blood leukocytes (neutrophils and monocytes), and macrophages in pathological conditions. MPO is capable of catalyzing hydrogen peroxide (H) ₂ O ₂ ) And chloride ion (Cl) ^- ) The reaction produces HOCl, and currently MPO is the only enzyme known to catalyze the generation of hypochlorous acid (HOCl) under physiological conditions. In addition to HOCl, MPO can also catalyze the production of other reactive oxygen/reactive nitrogen species (ROS/RNS), such as tyrosinyl and hydroxyl radicals (R: (R) (R)) ^· OH). Abnormal MPO expression produces excessive ROS/RNS, which can directly oxidize DNA, protein and lipidLarge molecules cause cell death and tissue damage. Many studies have shown that this is associated with the pathogenesis of many diseases, such as rheumatoid arthritis, atherosclerosis, alzheimer's disease, parkinson's disease and certain cancers, and MPO has been identified as one of the biomarkers for predicting and diagnosing cardiovascular and cerebrovascular diseases. Therefore, the detection of MPO activity in organisms is of great importance for understanding the pathological role of MPO and for the early diagnosis and treatment of diseases.

At present, the methods for directly detecting MPO activity are not many, and enzyme-linked immunosorbent assay (ELISA) is commonly used in clinic, and the detection principle of the method is based on the specific binding reaction between antigen and antibody to quantitatively detect MPO. Although the ELISA method has high sensitivity, high selectivity and good repeatability, the disadvantages of long time consumption, high cost and the like limit the wide application of the ELISA method. Fluorescent probes have attracted increasing attention due to their advantages of high selectivity and sensitivity, fast response times, and relatively simple instrumentation required for detection. Based on that MPO in organisms is the only enzyme which can catalyze and generate HOCl, a fluorescent probe based on HOCl response is developed, and the detection of the MPO activity in the organisms is realized. Compared with a commercial ELISA kit, the method can greatly save the detection time and reduce the cost, and is beneficial to the early diagnosis of MPO related diseases, so that the development of the fluorescent probe has very important significance.

Disclosure of Invention

The invention aims to provide a novel fluorescent probe for detecting myeloperoxidase as well as a preparation method and application thereof.

The fluorescent probe for detecting myeloperoxidase provided by the invention is a compound with a structure shown in formula I:

。

wherein:

(1) Z is O or S;

(2) R1, R4, R5, R6, R7 and R10 may each independently be selected from a hydrogen atom, a halogen or an alkyl group;

(3) R2, R3, R8 and R9 may each independently be selected from a hydrogen atom, an unsubstituted alkyl group, a phenyl-substituted alkyl group, an alkoxy group or a hydroxyalkoxy group;

(4) The alkyl group in the present invention means a saturated alkane, and includes a straight-chain or branched methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, an isopropyl group and a tert-butyl group.

The preparation method of the fluorescent probe provided by the invention has the following reaction route:

。

the method comprises the following specific steps:

(1) Preparation of intermediate B

After the compound A and alkali are mixed, a reducing agent is dripped to obtain an intermediate B, and the intermediate B can directly enter the next reaction without separation;

the base is selected from various organic bases and inorganic bases, preferably sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate. The reducing agent is selected from sodium hydrosulfite, vitamin C and ferrous ammonium sulfate, preferably sodium hydrosulfite. The solvent is preferably a mixed system of dichloromethane and water, and the reaction temperature is 20-80 ℃, preferably 40 ℃;

(2) Preparation of fluorescent Probe I

Adding triphosgene into a reaction system containing the compound B to prepare an intermediate C, wherein the intermediate C can be directly used for the next reaction without purification;

dissolving alkali, 4-Dimethylaminopyridine (DMAP) and ammonia water or substituted amino derivative in a solvent, dropwise adding a compound C, and reacting to obtain a compound I.

In the present invention, the base is selected from various organic bases and inorganic bases, and preferably sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate. The solvent is preferably dichloromethane, and the reaction temperature is-10-20 deg.C, preferably 0 deg.C.

In the invention, the compound with the structure I and hypochlorous acid have the following response mechanism:

。

the fluorescent probe (i.e. the compound having the structure of formula I) of the present invention does not have fluorescence, and generates an intermediate B in response to hypochlorous acid, and is further oxidized by hypochlorous acid to generate a compound A having strong fluorescence emission.

The fluorescent probe can be used for detecting MPO activity (in solution and animal living body level). The method comprises the following specific steps:

(1) Dissolving a fluorescent probe in an organic solvent to prepare 1 mM mother liquor, then diluting the mother liquor into a PBS (PBS) buffer solvent (10 mM, pH = 7.2) to prepare a 5 mu M solution, and responding to hypochlorous acid to detect the MPO activity;

(2) The fluorescent probe was dissolved in an organic solvent to prepare 1 mM stock solution, which was then injected 100. Mu.L into an animal model of inflammation for in vivo level measurement of MPO activity.

Here, the organic solvent is methanol, ethanol, N-dimethylformamide or the like.

The invention has the following effects:

(1) Fluorescent probes respond very rapidly to hypochlorous acid (< 10 s) and have low detection limits for detecting MPO activity (LOD = 4.2 mU/mL);

(2) The fluorescent probe has excellent selectivity on hypochlorous acid and does not respond to other common active oxygen/active nitrogen;

(3) The fluorescent probe can be applied to living body level and is used for detecting MPO activity in a rat arthritis model based on hypochlorous acid response.

Drawings

FIG. 1 shows a fluorescent probe FD-301 according to example 1 of the present invention ¹ H NMR Spectrum (400 MHz, DMSO-d) ₆ ）。

FIG. 2 shows a fluorescent probe FD-301 according to example 1 of the present invention ¹³ C NMR spectrum (100 MHz, DMSO-d) ₆ ）。

FIG. 3 shows the fluorescence spectrum of the fluorescent probe FD-301 of example 1 before and after response to hypochlorous acid. The concentration of FD-301 was 5. Mu.M, and the excitation wavelength of hypochlorous acid was 620 nm at a concentration of 15. Mu.M.

FIG. 4 is a graph showing the time dependence of the response of the fluorescent probe FD-301 and hypochlorous acid according to the present invention.

FIG. 5 shows the selective fluorescence spectrum of the fluorescent probe FD-301 of example 1 according to the present invention. FD-301 was at a concentration of 5. Mu.M, and ROS/RNS and hypochlorous acid were at different concentrations as shown in the figure. The comparative ROS/RNS from A to J are: t-BuOO ^• , KO ₂ , NO, ^• OH, ONOO ^- , ROO ^• , H ₂ O ₂ t-BuOOH, HOCl. The comparison is the fluorescence intensity at 686 nm emission with an excitation wavelength of 620 nm.

FIG. 6 is a fluorescence spectrum of the response of the fluorescent probe FD-301 according to the present invention to MPO (0, 1, 10, 50, 100 and 500 mU/mL) at various concentrations. FD-301 concentration was 5. Mu.M, H ₂ O ₂ The concentration of (2) was 10. Mu.M, and the excitation wavelength was 620 nm.

FIG. 7 is a standard curve for detecting MPO activity using the fluorescent probe FD-301 according to the present invention. FD-301 concentration was 5. Mu.M, H ₂ O ₂ With a concentration of 10 μm, a concentration of standard curve MPO of 10, 20, 30, 40 and 50 mU/mL, respectively, and an excitation wavelength of 620 nm.

FIG. 8 is a graph showing the fluorescence imaging of the fluorescent probe FD-301 according to the present invention in a mouse arthritis model.

FIG. 9 shows a fluorescent probe FD-302 according to example 2 of the present invention ¹ H NMR spectrum (400 MHz, DMSO-d) ₆ ）。

FIG. 10 shows fluorescence spectra of the fluorescent probe FD-302 according to example 2 of the present invention before and after response to hypochlorous acid. FD-302 was 5. Mu.M in concentration, and hypochlorous acid was 15. Mu.M in concentration, and the excitation wavelength was 620 nm.

FIG. 11 is a fluorescent spectrum of the response of the fluorescent probe FD-302 according to example 2 to different concentrations of MPO (0 and 50 mU/mL). FD-302 concentration was 5. Mu.M, H ₂ O ₂ The concentration of (2) was 10. Mu.M, and the excitation wavelength was 620 nm.

Detailed Description

Example 1

Preparing a compound FD-301 with a structure shown as formula I and detecting MPO activity:

。

(1) Preparation of Compound FD-301

Methylene blue (2.0 g, 6.25 mmol) was added to a three-neck flask and dissolved with 15mL of methylene chloride and 40mL of water. Sodium carbonate (2.65 g, 25.00 mmol) was added to the system, the system was stirred at 40 ℃, and sodium dithionite (3.26 g, 18.75 mmol) dissolved in 20mL of water was added dropwise to the system under nitrogen protection. After the addition is finished, the reaction is carried out for 20min, at the moment, the system is obviously layered, and the lower layer is yellow solution. The system was allowed to stand, 10mL of triphosgene (0.93 g, 3.13 mmol) dissolved in methylene chloride was added dropwise to the system, and after completion of the addition, the reaction mixture was transferred to room temperature and stirred for 1 hour. And (3) standing the system, sucking the lower clear solution out by using an injector after layering, dripping the lower clear solution into a mixed system mixed with 4-aminobenzoic acid hydrazide (181.32 mg, 1.2 mmol), triethylamine (0.5 mL, 3.6 mmol) and 5mL of dichloromethane, keeping the system in an ice water bath during the dripping process, transferring to room temperature after the dripping is finished, and stirring for reaction by TLC (thin layer chromatography) until the reaction is finished.

Insoluble matter in the system was removed by filtration, and the filtrate was poured into ice water and extracted with ethyl acetate (100 mL. Times.4). The organic phases were combined, washed with 200mL of X3 saturated brine and dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by silica gel column chromatography to give 100 mg of FD-301 as a pale green solid in 55% yield (calculated with 4-aminobenzoic acid hydrazide as reference). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.75 (s, 1H), 7.92 (s, 1H), 7.58 (d, J = 7.6 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H), 6.72-6.67 (m, 4H), 6.53 (d, J= 7.2 Hz, 2H), 5.69 (s, 2H), 2.89 (s, 12H). See fig. 1.

¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 166.04, 155.21, 152.07, 148.61, 132.85, 129.03, 128.01, 126.95, 119.1, 112.53, 111.29, 110.31, 40.25. See fig. 2.

(2) Performance test of fluorescent Probe FD-301

A. Response of FD-301 to hypochlorous acid

As shown in fig. 3, 5 μ M FD-301 buffer (10 mM pbs, ph = 7.2, 1% ethanol) system was non-fluorescent. However, the mixed system has strong fluorescence response after adding 15 μ M hypochlorous acid, and the fluorescence intensity at 686 nm is increased by 126 times.

As shown in FIG. 4, the concentration of FD-301 was 5. Mu.M, and the concentration of hypochlorous acid was 15. Mu.M, and the excitation wavelength was 620 nm. It can be observed that the fluorescence intensity at 686 nm when hypochlorous acid is added to the system increases rapidly with time at an excitation wavelength of 620 nm and reaches equilibrium within < 10s, indicating that the probe can respond rapidly to hypochlorous acid.

B. Selectivity of FD-301

As shown in FIG. 5, even the hypochlorous acid concentration in the system is only 5. Mu.M, the FD-301 strong fluorescence response can be induced, while other ROS/RNS cannot induce the change of the FD-301 fluorescence intensity even at the concentration of 20. Mu.M, such as t-BuOO ^• , KO ₂ , NO, ^• OH, ONOO ^- , ROO ^• , H ₂ O ₂ t-BuOOH, the results show that FD-301 has excellent selectivity.

C. FD-301 measurement of MPO Activity

As shown in FIG. 6, when the concentration of FD-301 was 5. Mu.M, H ₂ O ₂ At a concentration of 10. Mu.M, the fluorescent probe FD-301 equilibrated with increasing MPO concentration when the MPO concentration was 100 mU/mL. The excitation wavelength was 620 nm.

As shown in FIG. 7, the limit of detection LOD (3) was obtained from the MPO standard curveσ/k) = 4.2 mU/mL。

D. Application of FD-301 in mouse inflammation model

As shown in FIG. 8, an inflammatory response model was constructed using carrageenan on the right ankle of mice, and fluorescence imaging detected a 4-fold increase in fluorescence at the site of inflammation compared to normal ankle following a simultaneous injection of 100 μ L of 1 mM FD-301 on both sides. The obvious fluorescence change of the probe before and after identification can be applied to living bodies.

Example 2

Preparation of compound FD-302 having the structure of formula I and hypochlorous acid response:

。

(1) Preparation of Compound FD-302

Basic blue 3 (2.0 g, 5.56 mmol) was added to a three-necked flask and dissolved with 15mL of methylene chloride and 40mL of water. Sodium carbonate (2.65 g, 25.00 mmol) was added to the system, the system was stirred at 40 ℃, and sodium dithionite (3.26 g, 18.75 mmol) dissolved in 20mL of water was added dropwise to the system under nitrogen protection. After the addition is finished, the reaction is carried out for 20min, at the moment, the system is obviously layered, and the lower layer is yellow solution. The system was allowed to stand, 10mL of triphosgene (0.93 g, 3.13 mmol) dissolved in methylene chloride was added dropwise to the system, and after completion of the addition, the reaction mixture was transferred to room temperature and stirred for 1 hour. And (3) standing the system, sucking the lower clear solution out by using an injector after layering, dripping the lower clear solution into a mixed system mixed with 4-aminobenzoic acid hydrazide (162.3 mg, 1.07 mmol), triethylamine (0.5 mL, 3.6 mmol) and 5mL of dichloromethane, keeping the system in an ice water bath during dripping, transferring to room temperature after dripping, and stirring for reaction by TLC (thin layer chromatography) until the reaction is finished.

Insoluble matter in the system was removed by filtration, and the filtrate was poured into ice water and extracted with ethyl acetate (100 mL. Times.4). The organic phases were combined, washed with 200mL of X3 saturated brine and dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by silica gel column chromatography to give 100 mg of FD-302 as a pale green solid in a yield of 60% (calculated with 4-aminobenzoic acid hydrazide as a reference). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.37 (dd, J = 8.0, 9.2 Hz, 4H), 6.62 (d, J = 8.0 Hz, 2H), 6.37-6.43 (m, 4H), 6.06 (s, 2H), 3.29-3.33 (m, 8H), 1.07 (t, J= 6.6 Hz, 12H). See fig. 9.

(2) Performance test of fluorescent Probe FD-302

A. Response of FD-302 to hypochlorous acid

As shown in fig. 10, 5 μ M FD-302 buffer (10 mM pbs, ph = 7.2, 1% ethanol) system showed very weak fluorescence. However, after 15 μ M hypochlorous acid is added, the fluorescence of the mixed system is obviously enhanced, and the fluorescence intensity at 686 nm is increased by 6 times.

B. FD-302 measurement of MPO Activity

As shown in FIG. 11, when the concentration of FD-301 was 5. Mu.M, H ₂ O ₂ At a concentration of 10. Mu.M, the fluorescent probe FD-302 showed a significant increase in intensity in response to 50 mU/mL MPO as compared to the blank. The excitation wavelength was 620 nm.

Claims

1. A fluorescent probe for detecting myeloperoxidase activity, which is a compound having a structure represented by formula FD-301 or FD-302:

2. a method for preparing a fluorescent probe for detecting myeloperoxidase activity according to claim 1, wherein:

the synthetic route of FD-301 is as follows:

the method comprises the following specific steps: 2.0g of 6.25mmol of methylene blue are added to a three-necked flask and dissolved with 15mL of dichloromethane and 40mL of water; 2.65g and 25.00mmol of sodium carbonate are added into the system; stirring the system at 40 ℃, dropwise adding 3.26g of 18.75mmol of sodium hydrosulfite dissolved by 20mL of water into the system after the nitrogen protection; after the addition, the reaction is carried out for 20min, at this time, the system is obviously layered, and the lower layer is yellow solution; standing the system, and then dropwise adding 0.93g of triphosgene dissolved in 10mL of dichloromethane and 3.13mmol of triphosgene into the system; after the dripping is finished, transferring the mixture to room temperature and stirring the mixture for reaction for 1 hour; standing the system, sucking out the lower clear liquid by using an injector after layering, dripping into a mixed system mixed with 181.32mg, 1.2mmol, 4-aminobenzoic acid hydrazide, 0.5mL, 3.6mmol of triethylamine and 5mL of dichloromethane, keeping the system in an ice water bath during the dripping process, transferring to room temperature after the dripping is finished, and stirring for reaction until the reaction is finished;

filtering to remove insoluble substances, pouring the filtrate into ice water, and extracting with ethyl acetate; the organic phases were combined, washed with 200mL of X3 saturated brine and dried over anhydrous sodium sulfate; removing the solvent on a rotary evaporator, and purifying the remainder by silica gel column chromatography to obtain a light green solid product FD-301;

the synthetic route of FD-302 is as follows:

the preparation method comprises the following steps:

2.0g of 5.56mmol of basic blue 3 is added into a three-neck flask and dissolved by 15mL of dichloromethane and 40mL of water; adding 2.65g of 25.00mmol of sodium carbonate into the system, stirring the system at 40 ℃, and dropwise adding 3.26g of 18.75mmol of sodium hydrosulfite dissolved by 20mL of water into the system after the nitrogen protection; after the addition, the reaction is carried out for 20min, at this time, the system is obviously layered, and the lower layer is yellow solution; standing the system, then dropwise adding 0.93g of triphosgene dissolved in 10mL of dichloromethane and 3.13mmol of triphosgene into the system, and transferring the system to room temperature for stirring and reacting for 1h after the dropwise addition; standing the system, sucking out the lower clear liquid by using an injector after layering, dripping into a mixed system mixed with 162.3mg and 1.07mmol of 4-aminobenzoic acid hydrazide, 0.5mL and 3.6mmol of triethylamine and 5mL of dichloromethane, keeping the system in an ice water bath during the dripping process, and transferring to room temperature for stirring reaction after the dripping is finished until the reaction is finished;

filtering to remove insoluble substances, pouring the filtrate into ice water, and extracting with ethyl acetate; the organic phases were combined, washed with 200mL of X3 saturated brine and dried over anhydrous sodium sulfate; removing the solvent on a rotary evaporator, and purifying the residue by silica gel column chromatography to obtain a light green solid product FD-302.